Immune modulation with tlr9 agonists for cancer treatment

ABSTRACT

The present invention relates to methods for treating a tumor, including a metastatic tumor, with TLR9 agonist in combination with an immune checkpoint inhibitor therapy.

PRIORITY

This Application claims priority to, and the benefit of, U.S.Provisional Application No. 62/394,845 filed Sep. 15, 2016, and U.S.Provisional Application No. 62/486,738 filed Apr. 18, 2017, each ofwhich is hereby incorporated by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:105968-5001_Sequence_Listing, date recorded: Sep. 15, 2107; file size:26 KB).

FIELD

The invention relates to the field of oncology, and use of immunotherapyin the treatment of cancer.

BACKGROUND

Toll-like receptors (TLRs) are present on many cells of the immunesystem and are involved in the innate immune response. In vertebrates,this family consists of eleven proteins called TLR1 to TLR11 thatrecognize pathogen associated molecular patterns from bacteria, fungi,parasites, and viruses. TLRs are a key mechanism by which vertebratesrecognize and mount immune responses to foreign molecules and alsoprovide a link between the innate and adaptive immune responses. SomeTLRs are located on the cell surface to detect and initiate a responseto extracellular pathogens and other TLRs are located inside the cell todetect and initiate a response to intracellular pathogens.

TLR9 recognizes unmethylated CpG motifs in bacterial DNA and insynthetic oligonucleotides. While agonists of TLR9, and other TLRagonists, can initiate anti-tumor immune responses, TLR agonists canalso induce immune suppressive factors that may be counterproductive foreffective tumor responses.

There is a need for cancer immunotherapies that induce antitumorresponses, and keep the immune system productively engaged to improvethe overall response.

SUMMARY

In various aspects, the present invention provides a method for treatinga tumor, including, without limitation, metastatic melanoma, comprisingintratumorally administering an oligonucleotide TLR9 agonist (e.g.,IMO-2125 or other immunostimulatory oligonucleotides described herein)to a cancer patient in combination with immunotherapy with an immunecheckpoint inhibitor therapy, such as a therapy targeting CTLA-4,PD-1/PD-L1/PD-L2, TIM3, LAG3, and/or IDO. The TLR9 agonist uponintratumoral injection induces global increases in expression ofcheckpoint genes, including IDO1, PDL1, PD1, IDO2, CEACAM1, OX40, TIM3,LAG3, CTLA4, and OX40L. By altering immune signaling in the tumormicroenvironment, such changes in gene expression provide opportunitiesto improve responsiveness to checkpoint inhibitor therapy, including insome embodiments, a complete response. The invention further providesthe opportunity to balance anti-tumor responses with inhibitory signals,thereby also minimizing immune-related adverse events (irAEs) ofcheckpoint inhibitor therapy.

In various embodiments, the patient has a cancer that was previouslyunresponsive to, or had become resistant to, a checkpoint inhibitortherapy, such as anti-CTLA-4, anti-PD-1, or anti-PD-L1 and/or anti-PD-L2agent. The invention finds use for treating primary cancer or ametastatic cancer, including cancers that originate from skin, colon,breast, or prostate, among other tissues. In some embodiments, thecancer is progressive, locally advanced, or metastatic carcinoma. Insome embodiments, the cancer is metastatic melanoma.

In accordance with embodiments of the invention, the immunostimulatoryoligonucleotide (e.g., IMO-2125) is administered intratumorally.Intratumoral administration alters immune signaling in the tumormicroenvironment, priming the immune system for an effective anti-tumorresponse, while inducing changes that are compatible with more effectivecheckpoint inhibitor therapy. For example, the TLR9 agonist (e.g.,IMO-2125) may be administered intratumorally at from about 4 mg to about64 mg per dose, with from about 3 to about 12 doses being administeredover 10 to 12 weeks. For example, therapy may be initiated with 3 to 5weekly doses of IMO-2125, optionally followed by 3 to 8 maintenancecloses, which are administered about every three weeks.

During the regimen of IMO-2125 (or other TLR9 agonist), one or morecheckpoint inhibitor therapies are administered to take advantage of thechanges in immune signaling. In some embodiments, the patient receivesan anti-CTLA-4 agent (e.g., ipilimumab or tremelimumab) and/or ananti-PD-1 agent (e.g., nivolumab or pembrolizumab). The immunecheckpoint inhibitor can be administered parenterally, such as, in someembodiments, subcutaneously, intratumorally, intravenously. For example,in various embodiments the immune checkpoint inhibitor is administeredat a dose of from about 1 mg/kg to about 5 mg/kg intravenously. Theinitial dose of the immune checkpoint inhibitor can be administered atleast one week after the initial TLR9 agonist dose, for example in aboutweeks 2, 3 or 4. In some embodiments, the immunotherapy agent isadministered from about 2 to about 6 times (e.g., about 4 times,preferably every three weeks).

In some embodiments, IMO-2125 is administered intratumorally to ametastatic melanoma patient previously found to be unresponsive or onlypartially responsive to PD-1 blockade therapy. For example, IMO-2125 isadministered at a dose of from 4 to 32 mg per dose in weeks 1, 2, 3, 5,8, and 11, with ipilimumab i.v. at 3 mg/kg. Ipilimumab can beadministered every three weeks, beginning in week 2. Alternatively,pembrolizumab can be administered i.v. at 2 mg/kg every three weeksbeginning on week 2.

The present methods in various embodiments allow for a robust anti-tumorimmune response (which in some embodiments is a complete response), andwhich does not come at the expense of significant side effects, e.g.relative to side effects observed when one or more immunotherapies areused in the absence of the TLR9 agonist. Such side effects includecommonly observed immune-related adverse events that affect varioustissues and organs including the skin, the gastrointestinal tract, thekidneys, peripheral and central nervous system, liver, lymph nodes,eyes, pancreas, and the endocrine system; such as hypophysitis, colitis,hepatitis, pneumonitis, rash, and rheumatic disease (among others).

Other aspects and embodiments will be apparent from the followingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-H show tumor growth reduction in a CT26.CL25 tumor model withIMO-2125 monotherapy, Tumor volume for treated tumors and distant tumorsis shown.

FIGS. 2A-F show tumor infiltrating lymphocytes in tumor nodules from Day28 of the experiment shown in FIG. 1A-H. Magnification is ×400.

FIGS. 2G-H show FACS data that shows CD8⁺ T cells tumor infiltrationwith IMO-2125 monotherapy (0.5 mg/kg).

FIG. 3 shows assays to demonstrate specific cytotoxic T cell responsesto tumor antigens.

FIGS. 4A-C show a study design to evaluate the relationship ofintratumoral IMO-2125 antitumor activity and infiltrating CD4⁺ and CD8⁺T cells.

FIGS. 4D-E show the impact of CD4⁺ and CD8⁺ T cell depletionin treatedand distal tumors.

FIG. 5A shows a study design to evaluate the duration and specificity ofthe antitumor response induced by intratumoral IMO-2125 treatment.

FIG. 5B-C show the tumor growth of mice rechallenged with CT26 or A20and intratumoral IMO-2125.

FIGS. 6A-H show a tumor study in the A20 model comparing intratumor andsubcutaneous administration. FIGS. 6A-D show the study design and tumorkinetics while FIGS. 6E-H show the presence of tumor-infiltratinglymphocytes (TILS) and changes in gene expression of various checkpointgenes.

FIG. 7A shows a study design to evaluate the antitumor activity ofintratumoral IMO-2125 in combination with anti-CTLA-4 mAb on treatedtumors and systemic lung metastases.

FIGS. 7B-E show the anti-tumor effects of intratumoral IMO-2125 andanti-CTLA-4 mAb alone or in combination.

FIGS. 8A-E show anti-tumor activities of IMO-2125 and anti-CTLA-4 mAbalone or in combination on systemic lung metastasis. FIG. 8A showsnumber of lung tumor nodules in the various treatment groups and FIGS.8B-E show images of tumors in the various treatment groups (picturestaken on Day 13 after tumor implantation).

FIGS. 9A-D show TILs in metastatic nodules in the various treatmentgroups (CD3 IHC stain ×400).

FIGS. 10A-R show an evaluation of the antitumor activity of intratumoralIMO-2125 in combination with anti-PD-1 mAb in CT26 colon carcinoma tumormodel. FIG. 10A shows the study design. FIGS. 10B-I show the impact ofthe combination on tumor growth kinetics at treated and distal sites.FIG. 10J shows the impact of the combination on TILs (magnifications areshown). FIGS. 10K-R show checkpoint gene expression at treated anddistal sites after treatment with the combination.

FIGS. 11A-N show an evaluation of the antitumor activity of intratumoralIMO-2125 in combination with anti-PD-1 mAb on treated tumors andsystemic lung metastases in a B16 melanoma model. FIG. 11A shows thestudy design. FIGS. 11B-E show the impact of the combination on tumorgrowth kinetics at treated sites. FIGS. 11F-J show the combination'simpact on lung metastases. FIGS. 11K-N show histopathology of metastaticlung tumors (Circle: Large tumor nodule, Arrow: Small tumor nodule,Inset figures: HE stained (×40), and Large figures: CD3 stained (×400)).

FIG. 12 shows a study design to evaluate the antitumor activity ofintratumoral IMO-2125 in combination with an IDO-1 inhibitor on treatedtumors and systemic lung metastases.

FIGS. 13A-B show that intratumoral IMO-2125 anti-tumor activity ispotentiated by co-treatment with an IDO-1 inhibitor. FIG. 13A shows thenumber of lung tumor nodules in each treatment group. FIG. 13B shows thechange in tumor volume in each treatment group during the regimen.

FIG. 14 provides a closing overview in a study population of adults withunresectable or metastatic melanoma that progressed with ≥12 weeks ofPD-1-directed therapy (alone or in combination).

FIGS. 15A-C show dendritic cell maturation results pre-dose and 24 hourspost IMO-2125 injection for patient 003 (4 mg doses of IMO-2125).

FIGS. 15D-G show T-cell activation results in injected and distanttumors.

FIGS. 16A-C show expansion of top cell clones in distant lesions andinduction of IFN-γ for patient 003 (4 mg IMO-2125).

FIG. 17 shows tumor imaging pre- and post-therapy for patient 004 (8 mg2125).

DETAILED DESCRIPTION

In various aspects, the present invention provides a method for treatinga tumor, e.g. a metastatic tumor (including, without limitation,metastatic melanoma) comprising intratumorally administering anoligonucleotide TLR9 agonist (e.g., IMO-2125) to a cancer patient, incombination with immunotherapy with an immune checkpoint inhibitortherapy, such as a therapy targeting CTLA-4, PD-1/PD-L1/PD-L2, LAG3,TIM3, and/or IDO.

Exemplary immune checkpoint inhibitors include anti-PD-1, anti-PD-L1,anti-PD-L2, and anti-CTLA-4 agents. PD-1/PD-L1/PD-L2 antibodies inhibitthe interaction between PD-1 and its ligands (PD-L1 and PD-L2) on tumorcells to promote immune-mediated tumor destruction. CTLA-4 antibodiesblock the inhibitory signals to T-cells transmitted by CTLA-4. WhilePD-1 antibodies and CTLA-4 antibodies have emerged as importanttherapeutic options for a variety of cancers, many patients fail torespond. For example, some melanoma patients show no response toanti-PD-1 treatment, or even progress, after 12 weeks of treatment.Further, immune checkpoint blockade is associated with variousimmune-related adverse events, which can affect various tissues andorgans including the skin, the gastrointestinal tract, the kidneys,peripheral and central nervous system, liver, lymph nodes, eyes,pancreas, and the endocrine system. These immune-related adverse events(irAEs) can be severe, or even fatal, and may require discontinuation oftherapy. Examples of common irAEs are hypophysitis, colitis, hepatitis,pneumonitis, rash, and rheumatic disease.

Expression of the various immune checkpoint molecules on cells of theimmune system induces a complex series of events that determines whetheran immune response will be effective to combat the tumor, or otherwiseresult in immune tolerance. For example, increased expression of PD-1 ondendritic cells (DCs) promotes apoptosis of activated DCs, a criticalantigen presenting cell for anti-tumor immune responses. Park S J,Negative role of inducible PD-1 on survival of activated dendriticcells, J. Leukocyte Biology 95(4): 621-629 (2014). Further, expressionof IDO, PD-1-1, and CTLA-4 in the peripheral blood of melanoma patientsand can be associated with advanced disease and negative outcomes, andare interconnected, suggesting that multiple immune checkpoints mightrequire targeting to improve therapy in some cases. Chevolet I, et al.,Characterization of the in vivo immune networks of IDO, tryptophanmetabolism, PD-L1, and CTLA-4 in circulating immune cells in melanoma,Oncoimmunology 4(3) e982382-7 (2015).

The TLR9 agonist known as IMO-2125, which is described more fullyherein, upon intratumoral injection induces global increases inexpression of checkpoint genes, including IDO1 (5.3 fold), PDL1 (2.6fold), PD1 (2.5 fold), IDO2 (5.9 fold), CEACAM1 (2.1 fold), OX40 (1.4fold), TIM3 (2.9 fold), LAG (1.9 fold), CTLA4 (1.8 fold), and OX40L (1.5fold). See FIG. 6B. By altering immune signaling in the tumormicroenvironment, such changes in gene expression provide opportunitiesto improve responsiveness with checkpoint inhibitor therapy, and toachieve lasting anti-tumor immunity. Further, by targeting a singleimmune checkpoint molecule selected from the stronger inhibitory signalsof PD-1 or CTLA-4, in connection with the robust activation of antigenpresenting cells (e.g., DCs) and priming of T cells with IMO-2125, theinvention provides the opportunity to balance anti-tumor responses withinhibitory signals, thereby also minimizing irAEs of checkpointinhibitor therapy.

In various embodiments, the patient has a cancer that was previouslyunresponsive to, or had become resistant to, a checkpoint inhibitortherapy. For example, the cancer may be refractory or insufficientlyresponsive to an immunotherapy, such as anti-CTLA-4, anti-PD-1, oranti-PDL1 and/or PD-L2 agent, including for example, one or more ofipilimumab, tremelimumab, pembrolizumab and nivolumab. In variousembodiments, the cancer patient has progressed after or during treatmentwith an anti-CTLA-4, anti-PD-1, or anti-PD-L1 and/or PD-L2 agent,including for example, one or more of ipilimumab, tremelimumab,pembrolizumab and nivolumab (or agents related thereto) or shown noresponse to such treatment for at least about 12 weeks.

Other immune checkpoint inhibitors can be administered alone (e.g, inplace of) or in combination with anti-CTLA4 or anti-PD-1/anti-PD-L1,such as an inhibitor of IDO (e.g., IDO-1 or IDO-2), LAG3, TIM3, amongothers. These and other immune checkpoint inhibitors are described in US2016-0101128, which is hereby incorporated by reference in its entirety.For example, the patient may further receive a regimen of an IDO-1inhibitor such as Epacadostat.

In various embodiments, the cancer is a primary cancer or a metastaticcancer. A primary cancer refers to cancer cells at an originating sitethat become clinically detectable, and may be a primary tumor.“Metastasis” refers to the spread of cancer from a primary site to otherplaces in the body. Cancer cells can break away from a primary tumor,penetrate into lymphatic and blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasize) in normal tissueselsewhere in the body. Metastasis can be local or distant.

The cancer may have an origin from any tissue. The cancer may originatefrom skin, colon, breast, or prostate, and thus may be made up of cellsthat were originally skin, colon, breast, or prostate, respectively. Thecancer may also be a hematological malignancy, which may be lymphoma. Invarious embodiments, the primary or metastatic cancer is lung cancer,kidney cancer, prostate cancer, cervical cancer, colorectal cancer,pancreatic cancer, ovarian cancer, urothelial cancer, gastric/GEJcancer, head and neck cancer, glioblastoma, Merkel cell cancer, head andneck squamous cell carcinoma (HNSCC), non-small cell lung carcinoma(NSCLC), small cell lung cancer (SCLC), bladder cancer, prostate cancer(e.g. hormone-refractory) and hematologic malignancies.

In some embodiments, the cancer is progressive, locally advanced, ormetastatic carcinoma. In some embodiments, the cancer is metastaticmelanoma, and may be recurrent. In some embodiments, the metastaticmelanoma is stage III or IV, and may be stage IVA, IVB, or IVC. Themetastasis may be regional or distant.

IMO-2125 and related immunostimulatory oligonucleotides target TLR9, andact as TLR9 agonists to alter immune signaling in the tumormicroenvironment, and induce anti-tumor T cell responses.

In accordance with various embodiments, the TLR9 agonist comprises atleast two oligonucleotides linked together through their 3′ ends, so asto have multiple accessible 5′ ends. The linkage at the 3′ ends of thecomponent oligonucleotides is independent of the other oligonucleotidelinkages and may be directly via 3′ or 2′ hydroxyl groups, orindirectly, via a non-nucleotide linker or a nucleoside, utilizingeither the 2′ or 3′ hydroxyl positions of the nucleoside. Linkages mayalso employ a functionalized sugar or nucleobase of a 3′ terminalnucleotide. Exemplary TLR9 agonists are described in U.S. Pat. Nos.8,420,615, 7,566,702, 7,498,425, 7,498,426, 7,405,285, 7,427,405,including Tables 1 and 2A-2D of each, the entire contents of which arehereby incorporated by reference in their entireties.

In various embodiments, the TLR agonist is selected from:

5′-TCTGACG₁TTCT-X-TCTTG₁CAGTCT-5′ (SEQ ID NO: 1)5′-TCTGTCG₁TTCT-X-TCTTG₁CTGTCT-5′ (SEQ ID NO: 2)5′-TCG₁TCG₁TTCT-X-GTCTTG₁CTG₁CT-5′ (SEQ ID NO: 3)5′-TCG₁AACG₁TTCG₁-X-G₁CTTG₁CAAG₁CT-5′ (SEQ ID NO: 4)5′-CTGTCoG₂TTCTC-X-CTCTTG₂oCTGTC-5′ (SEQ ID NO: 5)5′-CTGTCG₂TTCTCo-X-oCTCTTG₂CTGTC-5′ (SEQ ID NO: 6)5′-TCG₁AACG₁TTCG₁-X-TCTTG₂CTGTCT-5′ (SEQ ID NO: 7)5′-TCG₁AACG₁TTCG₁-Y-GACAG₁CTGTCT-5′ (SEQ ID NO: 8)5′-CAGTCG₂TTCAG-X-GACTTG₂CTGAC-5′ (SEQ ID NO: 9)5′-CAGTCG₁TTCAG-X-GACTTG₁CTGAC-5′ (SEQ ID NO: 10)5′-TCG₁AACG₁TTCoG-Z-GoCTTG₁CAAG₁CT-5′ (SEQ ID NO: 11)5′-TCG₁AACG₁TTCG₁-Y₂-TCTTG₁CTGTCTTG₁CT-5′ (SEQ ID NO: 12)5′-TCG₁AACG₁TFCG₁-Y₂-TCTTG₁CTGUCT-5′ (SEQ ID NO: 13)5′-TCG₁AACG₁ToTCoG-m-GoCToTG₁CAAG₁CT-5′ (SEQ ID NO: 14)5′-TCG₁AACG₁TTCoG-Y₃-GACTTG₂CTGAC-5′ (SEQ ID NO: 15)5′-TCG₁AACG₁TTCG₁-Y₄-TGTTG₁CTGTCTTG₁CT-5′ (SEQ ID NO: 16)5′-TCG₂TCG₂TTU₁Y-M-YU₁TTG₂CTG₂CT-5′ (SEQ ID NO: 17)5′-CAGTCG₂TTCAG-Y₃-TCTTG₁CTGTCT-5′ (SEQ ID NO: 18)5′-TCG₁TACG₁TACG₁-X-G₁CATG₁CATG₁CT-5′ (SEQ ID NO: 19)5′-TCG₁AACG₁TTCG-Z-GCTTG₁CAAG₁CT-5′ (SEQ ID NO: 20)5′-TCG₁AACG₁TTCoG-Y₃-CTTG₂CTGACTTG₁CT-5′ (SEQ ID NO: 21)5′-TCG₁AACG₁oTTCG₁-X₂-G₁CTToG₁CAAG₁CT-5′ (SEQ ID NO: 22)5′-TCG₁AACG₁TTCG₁-Y₄-CATTG₁CTGTCTTG₁CT-5′ (SEQ ID NO: 23)5′-TCG₁AACG₁TTCG₁-m-G₁CTTG₁CAAG₁CT-5′ (SEQ ID NO: 24)5′-TCoG₁oAACoG₁TTCoG₁o-X₂-oG₁oCTTG₁oCAAoG₁oCT-5′ (SEQ ID NO: 25)5′-ToCG₁oAACoG₁TTCoG₁o-X₂-oG₁oCTTG₁oCAAoG₁CoT-5′ (SEQ ID NO: 26)5′-TCoG₁oAACoG₁TTCoG₁o-m-oG₁oCTTG₁oCAAoG₁oCT-5′ (SEQ ID NO: 27)5′-TCoG₂oAACoG₂TTCoG₂o-X₂-oG₂oCTTG₂oCAAoG₂oCT-5′ (SEQ ID NO: 28)5′-TCoG₁oAACoG₁TTCoGo-Z-oGoCTTG₁oCAAoG₁oCT-5′ (SEQ ID NO: 29) and5′-ToCG₁oAACoG₁TTCoGo-Z-oGoCTTG₁oCAAoG₁CoT-5′ (SEQ ID NO: 30),where G₁ is 2′-deoxy-7-deazaguanosine, G₂ is 2′-deoxy-arabinoguanosine;G, C, or U are 2′-O-methylribonucleotides; U₁ is 2′-deoxy-U; o is aphosphodiester linkage; X is a glycerol linker; X₂ is a isobutanetriollinker, Y is C3-linker; m is cis,trans-1,3,5-cyclohexanetriol linker; Y₂is 1,3-propanediol linker; Y3 is 1,4-butanediol linker; Y₄ is1,5-pentandiol linker; Z is 1,3,5-pentanetriol linker; and M iscis,cis-1,3,5-cyclohexanetriol linker.

In various embodiments, the TLR9 agonist is selected from5′-TCG₁AACG_(I)TTCG₁-X-G₁CTTG₁CAAG₁CT-5′ (SEQ ID NO:4),5′-CTGTCoG₂TTCTC-X-CTCTTG₂oCTGTC-5′ (SEQ ID NO:5), 5′-CTGTCG₂TTCTCo-X-oCTCTTG₂CTGTC-5′ (SEQ ID NO:6), 5′-TCG₁AACG₁TTCG₁-Y-TCTTG₂CTGTCT-5′ (SEQID NO:7), and 5′-TCG₁AACG₁TTCG₁-Y-GACAG₁CTGTCT-5′ (SEQ ID NO:8), whereinX is a glycerol linker, Y is a C3-linker, G₁ is2′-deoxy-7-deazaguanosine, G₂ is arabinoguanosine, and o is aphosphodiester linkage.

In various embodiments, the TLR9 agonist is5′-TCG₁AACG₁TTCG₁-X-G₁CTTG₁CAAG₁CT-5′ (SEQ ID NO:4), wherein X is aglycerol linker and G₁ is 2′-deoxy-7-deazaguanosine, otherwise known asIMO-2125.

Alternative TLR9 agonists are immune stimulatory oligonucleotidesdisclosed in U.S. Pat. No. 8,871,732, which is hereby incorporated byreference in its entirety. Such agonists comprise a palindromic sequenceof at least 8 nucleotides and at least one CG dinucleotide.

In accordance with embodiments of the invention, the immunostimulatoryoligonucleotide IMO-2125) is administered intratumorally. In someembodiments, the intratumoral administration is in a primary orsecondary tumor (e.g., metastatic melanoma lesion). Intratumoraladministration alters immune signaling in the tumor microenvironment,priming the immune system for an effective anti-tumor response, whileinducing changes that are compatible with more effective checkpointinhibitor therapy.

Illustrative dosage forms suitable for intratumoral administrationinclude solutions, suspensions, dispersions, emulsions, and the like.The TLR9 agonist may be provided in the form of sterile solidcompositions (e.g. lyophilized composition), which can be dissolved orsuspended in sterile injectable medium immediately before use. They maycontain, for example, suspending or dispersing agents known in the art.

In various embodiments, the TLR9 agonist is IMO-2125 and is administeredintratumorally at from about 4 mg to about 64 mg per dose, or in someembodiments from about 8 mg to about 64 mg per dose, or from about 12 mgto about 64 mg per dose, or from about 16 mg to about 64 mg per dose, orfrom about 20 mg to about 64 mg per dose. In some embodiments, IMO-2125is administered at from about 20 mg to about 48 mg per dose, or about 20mg to about 40 mg per dose. For example, in various embodiments,IMO-2125 is administered at about 4 mg, or about 8 mg, or about 12 mg,or about 16 mg, or about 20 mg, or about 24 mg, or about 28 mg, or about32 mg, or about 36 mg, or about 40 mg, or about 44 mg, or about 48 mg,or about 52 mg, or about 56 mg, or about 60 mg, or about 64 mg per dose,e.g. intratumorally.

In various embodiments, about 3 to about 12 doses of the TLR9 agonist(e.g. IMO-2125) are administered (e.g. about 3 doses, or about 4 doses,or about 5 doses, or about 6 doses, or about 7 doses, or about 8 doses,or about 9 doses, or about 10 doses, or about 11 doses, or about 12doses). In various embodiments, about 4 to about 8 doses areadministered over 10 to 12 weeks. In some embodiments, about 6 doses areadministered over 10 to 12 weeks. In some embodiments, therapy isinitiated with 3 to 5 weekly doses of IMO-2125, optionally followed by 3to 8 maintenance doses, which are administered about every three weeks.In some embodiments, an IMO-2125 dose is administered in weeks 1, 2, 3,5, 8, and 11. The IMO-2125 doses may be administered in the same ordifferent lesions.

During the regimen of IMO-2125 (or other TLR9 agonist), one or morecheckpoint inhibitor therapies are administered to take advantage of thechanges in immune signaling. The one or more checkpoint inhibitors canbe administered parenterally, including intravenously, intratumorally,or subcutaneously, among other methods. In some embodiments, the patientreceives an anti-CTLA-4 agent. For example, the anti-CTLA-4 agent may bean antibody that targets CTLA-4, for instance an antagonistic antibody.In various embodiments, the anti-CTLA-4 is ipilimumab (e.g. YERVOY,BMS-734016, MDX-010, MDX-101). In various embodiments, the anti-CTLA-4is tremelimumab (e.g. CP-675,206, MEDIMNIUNE). In other embodiments, theimmunotherapy agent is an anti-PD-1 agent. For example, the anti-PD-1agent may be an antibody that targets the PD-1, for instance, inhibitingthe interaction between PD-1 and PD-L1 (and/or PD-L2). In variousembodiments, the anti-PD-1 agent is nivolumab (ONO-4538/BMS-936558,MDX1106 or OPDIVO). In various embodiments, the anti-PD-1 agent ispembrolizumab (KEYTRUDA or MK-3475). In various embodiments, theanti-PD-1 agent is pidilizumab (CT-011 or MEDIVATION).

In some embodiments, the present immunotherapy agent is an anti-PD-L1and/or PD-L2 agent. For example, in various embodiments, the anti-PD-L1and/or PD-L2 agent is an antibody that targets PD-L1 and/or PD-L2, forinstance, inhibiting the interaction between PD-1 and PD-L1 and/orPD-L2. In various embodiments, the anti-PD-L1 and/or PD-L2 agent isatezolizumab (TECENTRIQ, ROCHE) BMS 936559 (BRISTOL MYERS SQUIBB), orMPDL328OA (ROCHE).

In various embodiments, the anti-CTLA-4, anti-PD-1, or anti-PD-L1 and/orPD-L2 agent (e.g. YERVOY, OPDIVO, or KEYTRUDA, or comparable agentsthereto) is administered at a dose of about 1 mg/kg, or about 2 mg/kg,or about 3 mg/kg, or about 4 mg/kg, or about 5 mg/kg, e.g.intravenously. For example, in some embodiments, the close of ananti-CTLA-4 agent, e.g. YERVOY, is about 3 mg/kg. For example, in someembodiments, the dose of an anti-PD-1 agent, e.g. OPDIVO, is about 3mg/kg. For example, in some embodiments, the dose of an anti-PD-1 agent,e.g. KEYTRUDA, is about 2 mg/kg. In various embodiments, the initialdose of the anti-CTLA-4, anti-PD-1, or anti-PD-L1 and/or PD-L2 agent(e.g. YERVOY, OPDIVO, or KEYTRUDA, or comparable agents thereto) isadministered at least one week after the initial TLR9 agonist dose, forexample in about weeks 2, 3 or 4.

In some embodiments, the immunotherapy agent is anti-CTLA4 (e.g.YERVOY), anti-PD-1 (e.g. OPDIVO or KEYTRUDA), or anti-PD-L1 and/oranti-PD-L2 agent, which is administered from about 2 to about 6 times(e.g. about 2 times, or about 3 times, or about 4 times, or about 5times, or about 6 times). In some embodiments, the immunotherapy agent,e.g. anti-CTLA-4 (e.g. YERVOY), anti-PD-1 (e.g. OPDIVO or KEYTRUDA), oranti-PD-L1 and/or PD-L2 agent is administered about 4 times.

In some embodiments, the immunotherapy agent is an anti-CTLA-4 agentsuch as YERVOY and is dosed at 3 mg/kg i.v. over about 90 minutes aboutevery 3 weeks. In some embodiments, the immunotherapy agent is ananti-PD-1 agent such as OPDIVO and is dosed at about 3 mg/kg i.v. overabout 60 minutes about every 2 weeks. In some embodiments, theimmunotherapy agent is an anti-PD-1 agent such as KEYTRUDA and is dosedat about 2 mg/kg i.v. over about 30 minutes about every 3 weeks.

In some embodiments, maintenance doses of the TLR9 agonist (e.g.IMO-2125), along with dosing of anti-CTLA-4, anti-PD-1, or anti-PD-L1and/or PD-L2 agent (e.g. YERVOY, OPDIVO, or KEYTRUDA, or comparableagents thereto) are administered about every 3 weeks.

In various embodiments, the present immunostimulatory oligonucleotidesallow for a dose reduction of the immunotherapy to about 10%, or about20%, or about 30%, or about 40%, or about 50%, or about 60%, or about70%, or about 80%, or about 90%, or about 100% of a monotherapy dose.For example, in some embodiments, an immunotherapy dose is about 0.1mg/kg, or about 0.3 mg/kg, or about 0.5 mg/kg, or about 0.7 mg/kg, orabout 1 mg/kg, or about 1.5 mg/kg, or about 2 mg/kg, or about 2.5 mg/kg,or about 3 mg/kg.

In some embodiments, IMO-2125 is administered intratumorally to ametastatic melanoma patient previously found to be unresponsive or onlypartially responsive to PD-1 blockade therapy. IMO-2125 is administeredat a dose of from 4 to 32 mg per dose (e.g., about 16 mg, about 20 mg,about 24 mg, about 28 mg, or about 32 mg) weeks 1, 2, 3, 5, 8, and 11,with ipilimumab i.v. at 3 mg/kg. Ipilimumab can be administered everythree weeks, beginning in week 2 (e.g., weeks 2, 5, 8, and 11).Alternatively, pembrolizumab can be administered i.v. at 2 mg/kg everythree weeks beginning on week 2 weeks 2, 5, 8, and 11).

In some embodiments, the patient further receives a regimen ofEpacadostat (an IDO-1 inhibitor), which may be administered at from 25mg to 300 mg orally, about twice daily. The regimen may be administeredfor about 5 day cycles. The first dose of Epacadostat may beadministered starting at about one week following the initial IMO-2125(or other TLR9 agonist) intratumoral injection.

In various embodiments, without wishing to be bound by theory, theinvention provides for a more balanced immune response in a cancerpatient, including cancer patients with advanced, metastatic disease.The combination therapy described herein can eliminate or reducedeficiencies that are observed in the respective monotherapies. Forexample, various patients are refractory to immunotherapies, or suchmonotherapies are hampered by extensive side effect profiles. Further asthe field is moving to combinations of immunotherapies (e.g. YERVOY andOPDIVO), such side effects are likely to be more problematic.

In various embodiments, the combination therapy allows for activationand/or maturation of dendritic cells, e.g. plasmacytoid dendritic cells,and modulates the tumor microenvironment (TME) in both treated anddistant tumors. For example, in various embodiments, the combinationtherapy provides for improvements in the amount or quality of TILSand/or CD8⁺ T cells to promote anti-tumor activities. For example,primed T cells are observed to invade both the proximal and distaltumors. Such primed T cells are suited for tumor invasion, particularlyat distal sites (e.g. secondary tumors), and, without wishing to bebound by theory, encounter a tumor environment that has reducedtolerance mechanisms in place. In various embodiments, the combinationtherapy provides for stimulation of interferons (e.g. IFN-α) and variousTh1 type cytokines (e.g. IFN-γ, IL-2, IL-12. and TNF-β).

The invention provides, in various embodiments, methods for treatingcancers, including metastatic cancers, in which the overall host immunemilieu is reengineered away from tumor tolerance. For example, a localTME is created that both disrupts pathways of immune tolerance andsuppression and allow for tumor regression. The present methods providein some embodiments, a TME capable of propagating a robust immuneresponse.

In various embodiments, a cancer patient's DCs are immature and unableto take up, process, or present antigens. These DCs may also beinhibited from migrating to regional lymph nodes or may inducetolerance, especially when presenting self-antigens. The cancerpatient's tumor site may also be infiltrated with regulatory T cellsthat are able to mediate suppression of antigen-primed T cells. Thehelper CD4 T cell response may also be skewed toward a Th2 phenotype,which inhibits the initiation of Th1 T cells and effective cellularimmunity. The tumor cells may express aberrant MHC class I molecules orβ2-microglobulin, resulting in inadequate antigen presentation and,thus, inefficient recognition of tumors by effector T cells. Finally,tumor cells and the surrounding stroma may release a number ofsuppressive cytokines, such as IL-6, IL-10, and TGF-β. This creates anenvironment that is not conducive to local immunity, which allows tumorcells to escape. In various embodiments, the present methods allow foran environment that is conducive to local immunity against tumors, e.g.,without limitation, maturation of DCs and/or reduction of regulatory Tcells and Th2 CD4 T cells.

In some embodiments, the combination therapy according to the inventionalters the balance of immune cells in favor of immune attack of a tumor.For instance, in some embodiments, the present methods shift the ratioof immune cells at a site of clinical importance, e.g. at the site ofagent administration or a distal site, in favor of cells that can killand/or suppress a tumor (e.g. T cells, cytotoxic T lymphocytes, T helpercells, natural killer (NK) cells, natural killer T (NKT) cells,anti-tumor macrophages (e.g. M1 macrophages), B cells, dendritic cells,or subsets thereof) and in opposition to cells that protect tumors (e.g.myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs);tumor associated neutrophils (TANs), M2 macrophages, tumor associatedmacrophages (TAMs), or subsets thereof). In some embodiments, thepresent methods increase a ratio of effector T cells to regulatory Tcells. In various embodiments, this altered balance of immune cells isaffected locally/proximally and/or systemically/distally. In variousembodiments, this altered balance of immune cells is affected in theTME.

Further, in various embodiments, the present methods allow for a robustanti-tumor immune response that does not come at the expense ofsignificant side effects (e.g., irAEs), e.g. relative to side effectsobserved when one or more immunotherapies are used in the absence of theTLR9 agonist.

For example, the combination therapy reduces one or more side effects ofan immunotherapy, e.g. an anti-CTLA-4, anti-PD-1, or anti-PD-L1 and/orPD-L2 agent, including for example, one or more of YERVOY, OPDIVO, andKEYTRUDA or agents related thereto. Such side effects include: fatigue,cough, nausea, loss of appetite, skin rash, itching pruritus, rash, andcolitis. In some embodiments, the side effects are intestinal problems(e.g. colitis) that can cause perforations in the intestines. Signs andsymptoms of the colitis may include: diarrhea or more bowel movementsthan usual; blood in the stools or dark, tarry, sticky stools; andabdominal pain or tenderness. In some embodiments, the side effects areliver problems (e.g. hepatitis) that can lead to liver failure. Signsand symptoms of hepatitis may include: yellowing of skin or the whitesof the eyes; dark urine; nausea or vomiting; pain on the right side ofthe stomach; and bleeding or bruising more easily than normal. In someembodiments, the side effects are skin problems that can lead to severeskin reactions. Signs and symptoms of severe skin reactions may include:skin rash with or without itching; sores in the mouth; and the skinblisters and/or peels. In some embodiments, the side effects are nerveproblems that can lead to paralysis. Symptoms of nerve problems mayinclude: unusual weakness of legs, arms, or face; and numbness ortingling in hands or feet. In some embodiments, the side effects arehormone gland problems (e.g. pituitary, adrenal, and thyroid glands).Signs and symptoms include: persistent or unusual headaches; unusualsluggishness; feeling cold all the time; weight gain; changes in mood orbehavior such as decreased sex drive, irritability, or forgetfulness;and dizziness or fainting. In some embodiments, the side effects areocular problems. Symptoms may include: blurry vision, double vision, orother vision problems; and eye pain or redness.

In some embodiments, patients experience fewer incidences of colitis,crohn's disease, or other GI involved irAE in accordance with thepresent invention.

In some embodiments, the patient achieves longer progression-freeinterval or longer survival (e.g., as compared to monotherapy), or insome embodiments, achieves remission or complete response. A completeresponse refers to the disappearance of all signs of cancer in responseto treatment.

This invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1: Anti-Tumor Effects of ImmunostimulatoryOligonucleotides (IMO-2125)

Immunomers were synthesized as is known in the art (see, e.g.,International Patent Publication No. WO 2016/057898, the entire contentsof which, inclusive of Example 1 and FIGS. 1 and 2 therein, are herebyincorporated by reference).

BALB/c mice (n=8 per group) were implanted s.c. with 2×10⁶ CT26.WT cellson right flank (Tumor 1) and 2×10⁶ CT126.CL25 cells on the left flank(Tumor 2). Treatment was initiated on Day 5 when tumor volume on rightflank reached 50 to 150 mm³.

Test compound was administered by intratumoral (i.t.) injection (100 μl)on right side tumor nodules (Tumor 1) only at Days 5, 8, 11 and 14.Tumor nodules were collected at Day 28. The text compounds were ControlDNA, IMO-2125: 0.5 mg/kg, IMO-2125: 2.5 mg/kg, and IMO-2125: 5 mg/kg. Asshown in FIGS. 1A-H, intratumoral IMO-2125 treatment led todose-dependent decreases in tumor volume in both treated and distanttumors. FIGS. 2A-H show tumor nodules collected on Day 28 after tumorimplantation. Immunohistochemical staining for CD3⁺ T lymphocyte surfacemarker. CD3⁺ cells stained brown color. While few CD3⁺ cells presentedinside tumor tissue bordering normal tissue from placebo-injected mice,a large number of CD3⁺ cells presented in the tumor tissue from micetreated with IMO-2125, 2.5 mg/kg. Results are shown in FIGS. 2A-F, whichdemonstrates inter alia, antitumor activity was associated withinduction of tumor infiltrating lymphocytes (TILS). FIGS. 2G-H show thatintratumoral IMO-2125 treatment increased infiltration of CD8⁺ T cellsin tumors.

Further, T cells from spleens of placebo—and IMO-2125 (2.5mg/kg)—treated tumor-bearing mice (n=3) were collected on Day 28.IFN-secreting ELISPOT was used for determining T cells specificallyagainst tumor internal antigen AH1 presented in both CT26.WT andCT26.CL25 and β-gal presented only in CT26.CL25. FIG. 3 shows thatintratumoral IMO-2125 treatment elicited specific cytotoxic T cellresponses to tumor antigens. In FIGS. 4A-E, the key role of CD8⁺ T cellsin treated and distal tumors is demonstrated.

FIGS. 5A-C show a study demonstrating intratumoral IMO-2125 induceddurable and tumor-specific immune memory. Six tumor-bearing mice (6 of9) whose tumors completely or partially regressed (<150 mm3) afterIMO-2125 (5 mg/kg, i.t.) treatments and 8 naïve BALB/c mice (n=8) ererechallenged on Day 33 with 1×106 C126 cells by s.c. injection atabdominal right and left flank. Naïve BALB/c mice inoculated same waywere used as tumor growth control. The mice that rejected CT26 tumorcell rechallenge (5 of 6) were then inoculated on Day 73 with 106syngeneic, non-organ-related B cell lymphoma A20 cells by s.c.inoculation at the upper back area. See the plan of FIG. 54. Results areshown in FIGS. 5B-C.

In FIGS. 6A-H, a study comparing intratumoral IMO-2125 is more effectivethan systemic (s.c.) treatment as demonstrated by antitumor activity inan A20 lymphoma model. BALB/c mice (n=10) were implanted s.c. with 3×10⁶A20 cells on the right and left flank. Treatment was initiated on day 8with intratumoral injection in the left flank with 2.5 mg/kg IMO-2125.IMO-2125 was given on days 8, 10, 12, and 14. Samples from placebo (PBS)control and IMO-2125 treated tumor-bearing mice were collected on day 21after tumor implantation. FIGS. 64-D show the study design and tumorkinetics. In FIGS. 6B-D, the tumor kinetics of subcutaneousadministration is slightly better than control while intratumoraladministration significantly slows tumor growth. FIGS. 6E-H show thepresence of TILs and changes in gene expression of various checkpointgenes. Importantly, IMO-2125 increased tumoral TILs and modulated tumorcheckpoint expression thereby sensitizing the TME for combination withone or more checkpoint inhibitors

Example 2: Anti-Tumor Effects of Combination Therapy of IMO-2125 and anAnti-CTL Antibody

FIGS. 7A-E show an evaluation of the antitumor activity of intratumoralIMO-2125 in combination with anti-CTLA-4 mAb on treated tumors andsystemic lung metastases. Study design is shown in FIG. 7A and resultsare shown in FIGS. 7B-E.

BALB/c mice were implanted s.c. with 2×10⁷ CT26 cells on right flank.The mice were than i.v. injected with 3×10⁶ CT26 cells to establish lungmetastases. Treatment was initiated on day 5. 2.5 mg kg IMO-2125 wasadministered intratumorally into CT26 solid tumors on the right flankand 10 mg/kg anti-CTLA-4 mAb was administered by interperitoneal (i.p.)injection. IMO-2125 and anti-CTLA-4 mAb were given either alone orco-administered on days 5, 6, 8 and 9. Lungs and T cells from spleens ofPBS control, IMO-4, anti-CTLA-4 mAb or IMO-2125 and anti-CTLA-4 mAbtreated tumor-bearing mice were collected.

Intratumoral IMO-2125 and anti-CTLA-4 mAb combination demonstratedimproved growth inhibition in treated tumors versus monotherapy witheither agent.

FIGS. 8A-E show anti-tumor activities of IMO-2125 and anti-CTLA-4 mAbalone or in combination on systemic lung metastasis.

FIGS. 9A-D show that intratumoral IMO-2125 and anti-CTLA-4 mAbcombination increased TILs in metastatic nodules.

The combination of intratumoral IMO-2125 and an anti-CTLA-4 mAb resultedin improved inhibition of tumor growth, regression of systemic lungmetastases and infiltration of TILS versus monotherapy with eitheragent. The effects were observed in directly treated tumors and systemiclung metastasis.

Example 3: Anti-Tumor Effects of Combination Therapy of IMO-2125 and anAnti-PD-1 Antibody

FIGS. 10A-R show an evaluation of the antitumor activity of intratumoralIMO-2125 in combination with anti-PD-1 mAb in CT26 colon carcinoma tumormodel. FIG. 10A shows the study design. BALB/c mice (n=8 per group) wereimplanted s.c. with 1×10⁷ murine colon carcinoma CT26 cells in rightflank (Tumor 1) and left flank (Tumor 2). Treatment was initiated on day7 when tumor volume on reached 200 to 300 mm³. 2.5 mg/kg IMO-2125 (50 μgin 100 μL PBS) was i.t injected at right tumor nodules and anti-PD-1 mAb(10 mg/kg, 200 μg/mouse) was administered by i.p. injection either aloneor co-administered on days 7, 8, 11 and 12 for total 4 times. Tumornodules were collected at day 14. Tumor growth inhibition, TILs andcheckpoint gene expression were evaluated at day 21. FIGS. 10B-I showthe impact of the combination on tumor growth kinetics at treated anddistal sites. The combination of IMO-2125 and anti-PD-1 demonstratedgrowth inhibition in both treated and distal sites that was superior toeither monotherapy. FIG. 10J shows the impact of the combination onTILs. intratumoral IMO-2125 and anti-PD-1 mAb combination increasedTILs. The PBS control group showed a few T cells (brown color); theIMO-2125 group showed large number of T cells; the PD-1 mAb group showedslightly increased T cells over PBS treated group; the combination groupshowed abundant T cells—more than IMO-2125 treated group (magnification:top row ×100, mid row ×200, bottom row ×400). FIGS. 10K-R showcheckpoint gene expression at treated and distal sites after treatmentwith the combination of IMO-2125 and anti-PD-1.

IMO-2124 and anti-PD-1 were tested in combination on treated tumors andsystemic lung metastases. See FIGS. 11A-N.

C57BL/6 mice (n=10) were implanted s.c. with 1×10⁷ B 16.F 10 cells inthe right flank (Tumor 1). The mice were than i. v. injected with 2×10⁶B16.F10 cells to establish lung metastases (Tumor 2), Treatment wasinitiated on day 5. 5 mg/kg IMO-2125 was administered intratumorallyinto B16 solid tumors on the right flank and 15 mg/kg anti-PD-1 mAb wasadministered by interperitoneal (i.p.) injection. IMO-2125 and anti-PD-1mAb were given either alone or co-administered on days 5, 6, 7, 8, and9. Samples from control, IMO-2125, anti-PD-1 mAb or IMO-2125 andanti-PD-1 mAb treated tumor-bearing mice were collected. FIG. 11A showsthe study design.

FIGS. 11B-E show the impact of the combination on tumor growth kineticsat treated sites.

FIGS. 11F-J show the combination's impact on lung metastases.Intratumoral injections of IMO-2125 in combination with anti-PD-1 mAbinduced potent systemic immune responses against disseminated lungmetastases

FIGS. 11K-N show histopathology of metastatic lung tumors (Circle: Largetumor nodule, Arrow: Small tumor nodule, Inset figures: HE stained(×40), and Large figures: CD3 stained (×400)), Treatment withintratumoral IMO-2125 and anti-PD-1 mAb combination led to decreasedlung tumor metastasis (inset and large figures) and creased TILs (largefigure).

Treatment with a combination of intratumoral IMO-2125 with an anti-PD-1antibody showed more potent antitumor activity than either agent alone.Antitumor activity was observed on treated as well as distant tumors.Infiltration levels of TILS increased in both treated and distanttumors. In preclinical models, IMO-2125 increased PD-L1 and othercheckpoint expression in the treated and distant tumors.

Example 4: Anti-Tumor Effects of Combination Therapy of IMO-2125 and anIDO-1 Inhibitor

FIG. 12 shows a study design to evaluate the antitumor activity ofintratumoral IMO-2125 in combination with an IDO-1 inhibitor on treatedtumors and systemic lung metastases in a muse model. Solid tumors andlung metastasis are implanted on Day 0 (solid tumor, 1×10⁷ CT26, s.c.,right flank; lung metastasis, 3×10⁶ CT26 i. v.), with IMO-2125 givenintratumorally (2.5 mg/kg) on Days 4, 5, 7, and 8. An IDO-1 inhibitor isadministered twice (75 mg/kg i.g.) on Days 4, 5, 7, and 8.

FIGS. 13A-B show that intratumoral IMO-2125 anti-tumor activity ispotentiated by co-treatment with an IDO-1 inhibitor. FIG. 13A shows thenumber of lung tumor nodules in each treatment group, showing theimprovement of IMO-2125 and IDO-1 inhibitor in comparison to each agentalone. FIG. 13B shows the change in tumor volume in each treatment groupduring the regimen.

Example 5: Study Population of Adults with Unresectable or MetastaticMelanoma that Progressed With >12 Weeks PD-1 Directed Therapy (Alone orin Combination)

FIG. 14 provides a dosing overview in a study population of adults withunresectable or metastatic melanoma that progressed with ≥12 weeks ofPD-1-directed therapy (alone or in combination). IMO-2125 wasadministered alone, intratumorally, in weeks 1 and 3. IMO-2125 wasadministered with ipilimumab or pembrolizumab in weeks 2, 5, 8. and 11.Administration of pembrolizumab continues every third week until time ofprogression.

FIGS. 15A-C show dendritic cell maturation results (CD1c, CD303, andHLA-DR expression) and pre-dose and 24 hours post i.t. IMO-2125injection for patient 003 (4 mg doses of IMO-2125; ipilimumab); andFIGS. 15D-G show T-cell activation results in injected and distanttumors.

FIGS. 16A-C show expansion of top cell clones in distant lesions, andcompares a non-responding patient with a responding patient (patient003, 4 mg IMO-2125, ipilimumab). The far right panel shows inductions ofIFN-γ for patient 003.

FIG. 17 shows tumor imaging pre- and post-therapy for patient 004 (8 mg2125, 3 mg ipilimumab). Injected and distant lesions are not visibleafter about 5 weeks of therapy.

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

1.-31. (canceled)
 32. A method for treating a cancer patient having kidney cancer, comprising intratumorally administering an initial dose of IMO-2125 TLR9 agonist to the kidney cancer patient, and administering at least one immune checkpoint inhibitor therapy that targets CTLA-4, PD-1, and/or PD-L1 to the patient about one or two weeks after the initial IMO-2125 TLR9 agonist dose.
 33. The method of claim 32, wherein the patient showed no response to prior treatment with PD-1 blockade therapy.
 34. The method of claim 33, wherein the prior PD-1 blockade therapy includes therapy with nivolumab or pembrolizumab.
 35. The method of claim 32, wherein the kidney cancer is a primary cancer.
 36. The method of claim 32, wherein the kidney cancer is a metastatic cancer.
 37. The method of claim 32, wherein the IMO-2125 TLR9 agonist is administered intratumorally at from about 4 mg to about 64 mg per dose.
 38. The method of claim 37, wherein the IMO-2125 TLR9 agonist is administered intratumorally at from about 4 mg to about 12 mg per dose.
 39. The method of claim 37, wherein the IMO-2125 TLR9 agonist is administered intratumorally at about 8 mg per dose.
 40. The method of claim 37, wherein the IMO-2125 TLR9 agonist is administered at from about 20 mg to about 64 mg per dose.
 41. (canceled)
 42. The method of claim 32, wherein about 3 to about 12 doses of IMO-2125 TLR9 agonist are administered.
 43. The method of claim 42, wherein about 4 to about 8 doses of IMO-2125 TLR9 agonist are administered over 10 to 12 weeks.
 44. The method of claim 43, wherein about 6 doses of IMO-2125 TLR9 agonist are administered over 10 to 12 weeks.
 45. The method of claim 42, wherein therapy is initiated with 3 to 5 weekly doses of IMO-2125 TLR9 agonist, followed by 3 to 9 maintenance doses administered about every three weeks.
 46. The method of claim 45, wherein the IMO-2125 TLR9 agonist is administered at least in weeks 1, 2, 3, 5, 8, and
 11. 47. The method of claim 32, wherein the patient receives an anti-CTLA-4 agent beginning on week 2 or week 3 after the initial IMO-2125 TLR9 agonist dose.
 48. The method of claim 47, wherein the anti-CTLA-4 agent is administered from 2 to 6 times, and optionally about 4 times.
 49. The method of claim 48, wherein the anti-CTLA-4 agent is administered at three-week intervals.
 50. The method of claim 47, wherein the anti-CTLA-4 agent is ipilimumab.
 51. The method of claim 32, wherein the patient receives an anti-PD-1 agent beginning on week 2 or week 3 after the initial IMO-2125 TLR9 agonist dose.
 52. The method of claim 51, wherein the anti-PD-1 agent is administered at least two times, and optionally about 4 times.
 53. The method of claim 52, wherein the anti-PD-1 agent is administered at at least three-week intervals.
 54. The method of claim 51, wherein the anti-PD-1 agent is pembrolizumab or nivolumab.
 55. The method of claim 32, wherein the at least one immune checkpoint inhibitor therapy is administered parenterally, and optionally by intravenous infusion, subcutaneous injection, or intratumoral injection.
 56. The method of claim 32, wherein the kidney cancer is renal cell carcinoma.
 57. The method of claim 32, wherein the patient receives an immune checkpoint inhibitor therapy that targets CTLA-4 and an immune checkpoint inhibitor therapy that targets PD-1.
 58. A method for treating kidney cancer in a patient, comprising administering an initial dose of IMO-2125 TLR9 agonist intratumorally to the kidney cancer patient previously found to be unresponsive or only partially responsive to PD-1 blockade therapy; the IMO-2125 TLR9 agonist being administered at a dose of from 4 to 32 mg per dose in at least weeks 1, 2, 3, 5, 8, and 11; and wherein ipilimumab and/or nivolumab is administered intravenously at at least three-week intervals beginning in week
 2. 59. The method of claim 58, wherein the ipilimumab and/or nivolumab is administered intravenously at from 2 to 4 mg/kg.
 60. The method of claim 58, wherein both ipilimumab and nivolumab are administered intravenously at at least three-week intervals beginning in week
 2. 61. A method for treating a kidney cancer patient, comprising: (i) intratumorally administering a dose of between 4 mg to 32 mg of IMO-2125 TLR9 agonist; (ii) administering an immune checkpoint inhibitor therapy selected from nivolumab, pembrolizumab, ipilimumab, tremelimumab, and combinations thereof; wherein the immune checkpoint inhibitor therapy is administered to the patient about one or two weeks after the initial IMO-2125 TLR9 agonist dose.
 62. The method of claim 61, comprising: (i) intratumorally administering the dose of between 4 mg to 32 mg of the IMO-2125 TLR9 agonist; (ii) administering ipilimumab; and (ii) administering nivolumab; wherein the ipilimumab and nivolumab are administered to the patient about one or two weeks after the initial IMO-2125 TLR9 agonist dose. 